The present invention relates to an apparatus for performing heat-exchanging, chemical reactions and for optically detecting a reaction product.
There are many applications in the field of chemical processing in which it is desirable to precisely control the temperature of reaction mixtures (e.g., biological samples mixed with chemicals or reagents), to induce rapid temperature changes in the mixtures, and to detect target analytes in the mixtures. Applications for such heat-exchanging chemical reactions may encompass organic, inorganic, biochemical and molecular reactions, and the like. Examples of thermal chemical reactions include isothermal nucleic acid amplification, thermal cycling nucleic acid amplification, such as the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, and more complex biochemical mechanistic studies that require complex temperature changes. Temperature control systems also enable the study of certain physiologic processes where a constant and accurate temperature is required.
One of the most popular uses of temperature control systems is for the performance of PCR to amplify a segment of nucleic acid. In this well known methodology, a DNA template is used with a thermostable DNA polymerase, nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to sequences that lie on opposite strands of the template DNA and which flank the segment of DNA that is to be amplified (xe2x80x9cprimersxe2x80x9d). The reaction components are cycled between a first temperature (e.g., 95xc2x0 C.) for denaturing double stranded template DNA, followed by a second temperature (e.g., 40-60xc2x0 C.) for annealing of primers, and a third temperature (e.g., 70-75xc2x0 C.) for polymerization. For some newer assays, the annealing and polymerization may be performed at the same temperature (e.g. 55-60xc2x0 C.), so that only two set point temperatures are required in each thermal cycle. Repeated cycling provides exponential amplification of the template DNA.
Nucleic acid amplification may be applied to the diagnosis of genetic disorders; the detection of nucleic acid sequences of pathogenic organisms in a variety of samples including blood, tissue, environmental, air borne, and the like; the genetic identification of a variety of samples including forensic, agricultural, veterinarian, and the like; the analysis of mutations in activated oncogenes, detection of contaminants in samples such as food; and in many other aspects of molecular biology. Polynucleotide amplification assays can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing and the analysis of mutations.
A preferred detection technique for chemical or biochemical analysis is optical interrogation, typically using fluorescence or chemiluminescence measurements. For ligand-binding assays, time-resolved fluorescence, fluorescence polarization, or optical absorption is often used. For PCR assays, fluorescence chemistries are often employed.
Conventional instruments for conducting thermal reactions and for optically detecting the reaction products typically incorporate a block of metal having as many as ninety-six conical reaction tubes. The metal block is heated and cooled either by a Peltier heating/cooling apparatus or by a closed-loop liquid heating/cooling system in which liquid flows through channels machined into the block. Such instruments incorporating a metal block are described in U.S. Pat. No. 5,038,852 to Johnson and U.S. Pat. No. 5,333,675 to Mullis.
These conventional instruments have several disadvantages. First, due to the large thermal mass of a metal block, the heating and cooling rates in these instruments are limited to about 1xc2x0 C./sec resulting in longer processing times. For example, in a typical PCR application, fifty cycles may require two or more hours to complete. With these relatively slow heating and cooling rates, some processes requiring precise temperature control are inefficient. For example, reactions may occur at the intermediate temperatures, creating unwanted and interfering side products, such as PCR xe2x80x9cprimer-dimersxe2x80x9d or anomalous amplicons, which are detrimental to the analytical process. Poor control of temperature also results in over-consumption of expensive reagents necessary for the intended reaction.
A second disadvantage of these conventional instruments is that they typically do not permit real-time optical detection or continuous optical monitoring of the chemical reaction. For example, in conventional thermal cycling instruments, optical fluorescence detection is typically accomplished by guiding an optical fiber to each of ninety-six reaction sites in a metal block. A central high power laser sequentially excites each reaction site and captures the fluorescence signal through the optical fiber. Since all of the reaction sites are sequentially excited by a single laser and since the fluorescence is detected by a single spectrometer and photomultiplier tube, simultaneous monitoring of each reaction site is not possible.
Some of the instrumentation for newer processes requiring faster thermal cycling times has recently become available. One such device is disclosed by Northrup et al. in U.S. Pat. No. 5,589,136. The device includes a silicon-based, sleeve-type reaction chamber that combines heaters, such as doped polysilicon for heating, and bulk silicon for convection cooling. The device optionally includes a secondary tube (e.g., plastic) for holding the sample. In operation, the tube containing the sample is inserted into the silicon sleeve. Each sleeve also has its own associated optical excitation source and fluorescence detector for obtaining real-time optical data. This device permits faster heating and cooling rates than the instruments incorporating a metal block described above. There are, however, several disadvantages to this device in its use of a micromachined silicon sleeve. A first disadvantage is that the brittle silicon sleeve may crack and chip. A second disadvantage is that it is difficult to micromachine the silicon sleeve with sufficient accuracy and precision to allow the sleeve to precisely accept a plastic tube that holds the sample. Consequently, the plastic tube may not establish optimal thermal contact with the silicon sleeve.
Another instrument is described by Wittwer et al. in xe2x80x9cThe LightCycler(trademark): A Microvolume Multisample Fluorimeter with Rapid Temperature Controlxe2x80x9d, BioTechniques, Vol. 22, pgs. 176-181, January 1997. The instrument includes a circular carousel for holding up to thirty-two samples. The temperature of the samples is controlled by a central heating cartridge and a fan positioned in a central chamber of the carousel. In operation, the samples are placed in capillaries which are held by the carousel, and a stepper motor rotates the carousel to sequentially position each of the samples over an optics assembly. Each sample is optically interrogated through a capillary tip by epi-illumination. This instrument also permits faster heating and cooling rates than the metal blocks described above. Unfortunately, this instrument is not easily configured for commercial, high throughput diagnostic applications.
The present invention overcomes the disadvantages of the prior art by providing an improved apparatus for thermally controlling and optically interrogating a reaction mixture. In contrast to the prior art instruments described above, the apparatus of the present invention permits extremely rapid heating and cooling of the mixture, ensures optimal thermal transfer between the mixture and heating or cooling elements, provides real-time optical detection and monitoring of reaction products with increased detection sensitivity, and is easily configured for automated, high throughput applications. The apparatus is useful for performing heat-exchanging chemical reactions, such as nucleic acid amplification.
In a preferred embodiment, the apparatus includes a reaction vessel having a chamber for holding the mixture. The vessel has a rigid frame defining the side walls of the chamber, and at least one flexible sheet attached to the rigid frame to form a major wall of the chamber. The rigid frame further includes a port and a channel connecting the port to the chamber to permit easy filling, sealing, and pressurization of the chamber. The apparatus also includes at least one thermal surface for contacting the flexible major wall of the chamber. The apparatus further includes a device for increasing the pressure in the chamber. The pressure increase in the chamber is sufficient to force the flexible major wall to contact and conform to the thermal surface, thus ensuring optimal thermal conductance between the thermal surface and the chamber. The apparatus also includes one or more thermal elements (e.g., a heating element, thermoelectric device, heat sink, fan, or peltier device) for heating or cooling the thermal surface to induce a temperature change within the chamber.
In the preferred embodiment, the reaction vessel includes first and second flexible sheets attached to opposite sides of the rigid frame to form opposing major walls of the chamber. In this embodiment, the apparatus includes first and second thermal surfaces formed by first and second opposing plates positioned to receive the chamber of the vessel between. When the pressure in the chamber is increased, the flexible major walls expand outwardly to contact and conform to the inner surfaces of the plates. A resistive heating element, such as a thick or thin film resistor, is coupled to each plate for heating the plates. In addition, the apparatus includes a cooling device, such as a fan, for cooling the plates. Each of the plates is preferably constructed of a ceramic material and has a thickness less than or equal to 1 mm for low thermal mass. In particular, it is presently preferred that each of the plates have a thermal mass less than about 5 J/xc2x0 C., more preferably less than 3 J/xc2x0 C., and most preferably less than 1 J/xc2x0 C. to enable extremely rapid heating and cooling rates.
The apparatus also preferably includes a support structure for holding the plates in an opposing relationship to each other. In the preferred embodiment, the support structure comprises a mounting plate having a slot therein, and spacing posts extending from the mounting plate on opposite sides of the slot. Each of the spacing posts has indentations formed on opposite sides thereof for receiving the edges of the plates. Retention clips hold the edges of the plates in the indentations formed in the spacing posts. The slot in the mounting plate enables insertion of the vessel between the plates.
The pressurization of the chamber ensures that the flexible major walls of the vessel are forced to contact and conform to the inner surfaces of the plates, thus. guaranteeing optimal thermal contact between the major walls and the plates. In the preferred embodiment, the device for increasing pressure in the chamber comprises a plunger which is inserted into the channel to compress gas in the vessel and thereby increase pressure in the chamber. The plunger preferably has a pressure stroke in the channel sufficient to increase pressure in the chamber to at least 2 psi of above the ambient pressure external to the vessel, and more preferably to a pressure in the range of 8 to 15 psi above the ambient pressure. In the preferred embodiment, the length of the pressure stroke is controlled by one or more pressure control grooves formed in the inner surface of the frame that defines the channel. The pressure control grooves extend from the port to a predetermined depth in the channel to allow gas to escape from the channel and thereby prevent pressurization of the chamber until the plunger reaches the predetermined depth. When the plunger reaches the predetermined depth, it establishes a seal with the walls of the channel and begins the pressure stroke. The pressure control grooves provide for highly controllable pressurization of the chamber and help prevent misalignment of the plunger in the channel.
The reaction vessel may be filled and pressurized manually by a human operator, or alternatively, the apparatus may include an automated machine for filling and pressurizing the vessel. In the automated embodiment, the apparatus preferably includes a pick-and-place machine having a pipette for filling the vessel and having a machine tip for inserting the plunger into the channel after filling. The plunger preferably includes a cap having a tapered engagement aperture for receiving and establishing a fit with the machine tip, thereby enabling the machine tip to pick and place the plunger into the channel.
In a second embodiment of the invention, the pressurization of vessel is performed by a pick-and-place machine having a machine head for addressing the vessel. The machine head has an axial bore for communicating with the channel. The pick-and-place machine also includes a pressure source in fluid communication with the bore for pressurizing the chamber of the vessel through the bore. In this embodiment, the apparatus also preferably includes a disposable adapter for placing the bore in fluid communication with the channel. The adapter is sized to be inserted into the channel such that the adapter establishes a seal with the walls of the channel. The disposable adapter preferably includes a valve (e.g., a check valve) for preventing fluid from escaping from the vessel.
In a third embodiment of the invention, the device for increasing pressure in the chamber comprises an elastomeric plug which is inserted into the channel, and a needle inserted through the plug for injecting fluid into the vessel. The needle may be used to inject the reaction mixture into the chamber, followed by air or another suitable gas to increase pressure in the chamber. The reaction vessel may be filled and pressurized in this manner by a human operator, or alternatively, the apparatus may include an automated machine for filling and pressurizing the chamber. In the automated embodiment, the apparatus includes a machine for inserting the needle through the plug, and the machine includes a pressure source for injecting fluid into the vessel through the needle.
In a fourth embodiment of the invention, the device for pressurizing the chamber comprises a platen for heat sealing a film or foil to the vessel. The foil is preferably sealed to the portion of the frame defining the port. Heat sealing the film or foil to the vessel seals the port and collapses an end of the channel to reduce the volume of the vessel and thereby increase pressure in the chamber. The reaction vessel may be heat sealed in this manner by a human operator, or alternatively, the apparatus may include an automated machine, e.g. a press, for sealing the vessel.
The apparatus of the present invention permits real-time monitoring and detection of reaction products in the vessel with improved optical sensitivity. In the preferred embodiment, at least two of the side walls of the chamber are optically transmissive and angularly offset from each other, preferably by an angle of about 90xc2x0. The apparatus further comprises an optics system for optically interrogating the mixture contained in the chamber through the optically transmissive side walls. The optics system includes at least one light source for exciting the mixture through a first one of the side walls, and at least one detector for detecting light emitted from the chamber through a second one of the side walls.
Optimum optical sensitivity may be attained by maximizing the optical sampling path length of both the light beams exciting the labeled analytes in the reaction mixture and the emitted light that is detected. The thin, wide reaction vessel of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume. In particular, the vessel is preferably constructed such that the ratio of the width of the chamber to the thickness of the chamber is at least 4:1, and such that the chamber has a thickness in the range of 0.5 to 2 mm. These parameters are presently preferred to provide a vessel having a relatively large average optical path length through the chamber, while still keeping the chamber sufficiently thin to allow for extremely rapid heating and cooling of the reaction mixture.
The apparatus of the present invention may be configured as a small hand-held instrument, or alternatively, as a large instrument with multiple reaction sites for simultaneously processing hundreds of samples. In high throughput embodiments, the plates, heating and cooling elements, and optics are preferably disposed in a single housing to form an independently controllable, heat-exchanging module with detection capability. The apparatus includes a base instrument for receiving a plurality of such modules, and the base instrument includes processing electronics for independently controlling the operation of each module. Each module provides a reaction site for thermally processing a sample contained in a reaction vessel and for detecting one or more target analytes in the sample. The apparatus may also include a computer for controlling the base instrument.